This invention features methods and apparatus for performing nucleic acid hybridization and amplification processes on a support. Such methods and apparatus are useful for synthesizing nucleic acid and detecting target nucleic acid for diagnostics and therapeutics.
The following definitions are provided to facilitate an understanding of the present invention. The term xe2x80x9cbiological binding pairxe2x80x9d as used in the present application refers to any pair of molecules which exhibit natural affinity or binding capacity. For the purposes of the present application, the term xe2x80x9cligandxe2x80x9d will refer to one molecule of the biological binding pair and the term xe2x80x9cantiligandxe2x80x9d or xe2x80x9creceptorxe2x80x9d will refer to the opposite molecule of the biological binding pair. Two complementary strands of nucleic acid are biological binding pairs. One of the strands is designated the ligand and the other strand is designated the antiligand. However, biological binding pairs may also comprise antigens and antibodies, drugs and drug receptor sites and enzymes and enzyme substrates.
The term xe2x80x9cprobexe2x80x9d refers to a ligand of known qualities capable of selectively binding to a target antiligand. As applied to nucleic acids, the term xe2x80x9cProbexe2x80x9d refers to a strand of nucleic acid having a base sequence complementary to a target base sequence. Typically, the probe is associated with a label to identify a target base sequence to which the probe binds, or the probe is associated with a support to bind to and capture a target base sequence. The term xe2x80x9cprimerxe2x80x9d is used to refer to nucleic acid having a base sequence complementary to a target base sequence, which upon nucleic acid hybridization is used to promote a reaction. These reactions usually involve enzymes called polymerases and transcriptases.
The term xe2x80x9clabelxe2x80x9d refers to a molecular moiety capable of detection including, by way of example, without limitation, radioactive isotopes, enzymes, luminescent agents, dyes and detectable intercalating agents. The term xe2x80x9cagentsxe2x80x9d is used in a broad sense, in reference to labels, and includes any molecular moiety which participates in reactions which lead to a detectable response. The term xe2x80x9ccofactorxe2x80x9d is used broadly to include any composition which participates in reactions with a label agent.
The term xe2x80x9csupportxe2x80x9d refers to conventional supports such as beads, particles, dipsticks, fibers, filters, membranes and silane or silicate supports such as glass.
The term xe2x80x9camplifyxe2x80x9d is used in the broad sense to mean creating an amplification product which may include, by way of example, additional target molecules, or target-like molecules or molecules complementary to the target molecule, which molecules are created by virtue of the presence of the target molecule in the sample. In the situation where the target is a nucleic acid, an amplification product can be made enzymatically with D-RA or RNA polymerases or transcriptases.
Genetic information is stored in living cells in threadlike molecules of deoxyribonucleic acid (DNA). In vivo, the DNA molecule is a double helix, each strand of which is a chain of nucleotides. Each nucleotide is characterized by one of four bases: adenine (A), guanine (G), thymine (T), and cytosine (C). The bases are complementary in the sense that due to the orientation of functional groups certain base pairs attract and bond to each other through hydrogen bonding. Adenine in one stand of DNA pairs with thymine in an opposing complementary stand. Guanine in one strand of DNA pairs with cytosine in an opposing complementary strand. In ribonucleic acid (RNA), the thymine base is replaced by uracil (U) which pairs with adenine in an opposing complementary strand.
DNA consists of covalently linked chains of deoxribonucleotides and RNA consists of covalently linked chains of ribonucleotides. The genetic code of a living organism is carried upon DNA in the sequence of the base pairs. Proteins are made or expressed by living organisms in a process in which a DNA sequence is transcribed to a RNA sequence and the RNA sequence translated into proteins.
Each nucleic acid is linked by a phosphodiester bridge between the five prime hydroxyl group of the sugar of one nucleotide and the three prime hydroxyl group of the sugar of an adjacent nucleotide. Each linear strand of naturally occurring DNA or RNA has one terminal end having a free five prime hydroxyl group and another terminal end having a three prime hydroxyl group. The terminal ends of polynucleotides are often referred to as being five prime (5xe2x80x2) termini or three prime (3xe2x80x2) termini in reference to the respective free hydroxyl soup. Complementary strands of DNA and RNA form antiparallel Complexes in which the 3xe2x80x2 terminal end of one strand is oriented to the 5xe2x80x2 terminal end of the opposing strand.
Nucleic acid hybridization assays detect the tendency of pairs of nucleic acid strands to pair with greatest stability if they contain regions of complementary sequence. Each pair of complementary nucleotides, between two strands, increases the stability of pairing between a biological binding pair formed between the two nucleic acids. DNA segments isolated from a growing organism are generally duplex DNA, a pair of perfectly complementary strands whose pairing is very stable. The term xe2x80x9chybridizexe2x80x9d refers to imposing conditions which promote such pairing. The term xe2x80x9cdenaturexe2x80x9d refers to imposing conditions which discourage such pairing. These conditions are imposed by adjusting ionic strength, pH or temperature.
Polymerases and transcriptases are enzymes which, in the presence of appropriate reaction conditions, produce a complementary copy of a strand of DNA or RNA. The strand that is copied is called the template DNA or RNA.
A polymerase chain reaction, PCR, uses a pair of nucleic acid primers to synthesize copies of target nucleic acid. One primer hybridizes to a target sequence on a first strand, and a second primer hybridizes to a second target sequence on the second strand. This permits the amplification product directed by one of the pair of primers to serve as a template for synthesis directed by the second member of the pair of primers. PCR is carried out using a solution containing both members of the pair of primers and a polymerase capable of withstanding conditions required to denature paired strands of DNA.
The identification of unique DNA or RNA sequence or specific genes within the total DNA or RNA extracted from tissue or culture samples may indicate the presence of physiological or pathological conditions. In particular, the identification of unique DNA or RNA sequences or specific genes, within the total DNA or RNA extracted from human or animal tissue, may indicate the presence of genetic diseases or conditions such as sickle cell anemia, tissue compatibility, cancer and precancerous states, or bacterial or viral infections. The identification of unique DNA or RNA sequences or specific genes within the total DNA or RNA extracted from bacterial cultures or tissue containing bacteria may indicate the presence of antibiotic resistance, toxins, viruses, or plasmids, or provide identification between types of bacteria.
Thus, nucleic acid hybridization assays have great potential in the diagnosis and detection of disease. Further potential exists in agricultural and food processing where nucleic acid hybridization assays may be used to detect plant pathogenesis or toxin-producing bacteria.
Much research is presently directed to identifying the nucleic acid sequences which define organisms. An initial step in the process is the identification of regions within the nucleic acid, a process known as mapping. These regions may be subjected to further sequencing. Both the mapping process and the sequencing process are slow and tedious.
The present invention features methods, articles of manufacture and devices for forming an amplification product in the presence of a first nucleic acid having a target sequence. The methods, articles of manufacture and devices feature the amplification of a first nucleic-acid without the use of solution base primer pairs. The method, articles of manufacture and instruments allow the performance of multiple simultaneous amplification reactions for rapid analysis of nucleic acids. The amplification reactions do not require the presence of an external reaction chamber, or the presence or use of gels for the analysis of the amplification product.
One embodiment of the present invention features a method for forming an amplification product in the presence of a first nucleic acid having a first target sequence. The method comprises the steps of forming an immersion product comprising a sample potentially containing the first nucleic acid, and a support. The support has a second nucleic acid having a sequence complementary to the target sequence. The second nucleic acid is covalently linked to the support. The method further comprises the step of forming a hybridization product comprising the first nucleic acid and the second nucleic acid, in the event the first nucleic acid is present in the sample. The hybridization product is formed by imposing hybridization conditions on the immersion product. The method further comprises the step of forming a first amplification product comprising a nucleic acid complementary to the first nucleic acid covalently extending from said second nucleic acid.
Preferably, the first nucleic acid comprises a second target sequence and the support comprises a third nucleic acid homologous to the second target sequence. Preferably, the method further comprises the step of releasing the first nucleic acid from the second nucleic acid by imposing the denaturation conditions on the immersion product. Release of the first nucleic acid allows the first nucleic acid to participate in further hybridization reactions.
Preferably, the release of the first nucleic acid also allows the amplification product to participate in further hybridization reactions to form a second hybridization product.
Preferably, the method further comprises imposition of a second step of hybridization conditions to form at least one second hybridization product. The second hybridization product comprises the first amplification product and a third nucleic acid or the first nucleic acid and a further second nucleic acid. Preferably, upon imposition of second hybridization conditions on the immersion product, the third nucleic acid forms a second hybridization with the first amplification product.
The formation of the second hybridization product allows the additional step of forming a second amplification product comprising nucleic acid complementary to the first amplification product covalently extending from the third nucleic acid. Thus, the first nucleic acid and the first and second amplification products, are capable of participating in a plurality of hybridization and amplification processes, limited only by the initial presence of the first nucleic acid and second and third nucleic acids initially present. Preferably, a plurality of second and third nucleic acids are covalently linked to the support to provide a plurality of first and second amplification products.
Preferably, the second nucleic acid and the third nucleic acid have positions on the support, which are spaced a distance less than the length of the first nucleic acid to allow an amplification product to form between the second and third nucleic acids. Preferably, a plurality of second and third nucleic acids have positions on the support, which are spaced a distance less than the length of the first nucleic acid, to form a plurality of first and second amplification products.
Preferably, the method further comprises the step of monitoring the support for the presence of one or more amplification products which one or more amplification products are indicative of the presence of one or more target sequence and which absence is indicative of the absence of a target sequence. The formation of a plurality of first and second amplification products allows the detection of extremely small numbers of first nucleic acid having target sequence.
Preferably, the support is epoxy silane derivatized silica. Supports may be filters, fibers, membranes, beads, particles, dipsticks, sheets, rods and the like. Preferably, the support has a composition of plastic, such as nylon or latex for beads, particles, dipsticks and the like; or glass, in the form of glass fiber, glass sheets, beads, rods, dipsticks; or metal, in the form of magnetic particles and the like. A preferred support comprises a sheet which has surfaces with alignment features to allow the precise positioning of the second nucleic acid and third nucleic acids, to define areas of the support directed to a first pair of target sequences and other areas directed to a second pair of target sequences. These areas are preferably arranged in a grid type pattern of pixels.
Preferably, the second nucleic acid is covalently bonded to a hexaethylene glycol functional group which functional group is covalently bonded to the support. However, other functional groups can be used to covalently bond DNA with a support. Preferably, the second nucleic acid is covalently bonded to the hexaethylene glycol functional group to a 5xe2x80x2 amino group. The hexaethylene glycol functional group positions the second nucleic acid away from the support to allow the second nucleic acid to interact with the first nucleic acid and enzymes used to form the amplification product.
Preferably, the first nucleic acid has a size of approximately 1 to 10 kb. Larger nucleic acids can be readily digested by enzymes or mechanically fragmented. Preferably, the second and third nucleic acid have a density or concentration on the support to allow spacing between such second and third nucleic acid less than the size of the first nucleic acid. Thus, the size of the first nucleic acid cooperates with the density concentration or spacing of second and third nucleic acids to allow amplification products to form there between.
As used herein, the term xe2x80x9cimmersion productxe2x80x9d refers to a support that is covered with a sample and other reagents about the nucleic acids covalently bonded to its surface. By way of example, making an immersion product may comprise placing a dipstick into a solution or placing beads in a solution, or wetting a slide or glass surface with a solution.
The term xe2x80x9chybridization productxe2x80x9d refers to the product of a hybridization reaction. The term xe2x80x9camplification productxe2x80x9d refers to a molecule or part of a molecule which has been made or extended by virtue of another molecule being present. Preferably, the amplification product is formed by imposing amplification conditions on the immersion product. Amplification conditions comprise applying a thermal stable polymerase nucleotides and other necessary reagents for a polymerase reaction to the hybridization product under conditions of temperature, ionic strength, and pH to support a polymerase reaction. As used herein, the term xe2x80x9capplyingxe2x80x9d means contacting or placing in proximity of an object in a manner such that the subject may act upon the object in the intended manner.
Preferably, the amplification product incorporates a label capable of detection. Preferred labels include radioisotopes, and chemiluminescent, luminescent and fluorescent agents and cofactors. However, where the amplification product participates in hybridization reactions to form a further hybridization product, such product can be detected with intercalating agents. In the alternative, the amplification product can be detected by a fourth nucleic acid probe which probe is complementary or homologous to a third target sequence derived from the first nucleic acid and present on one or more. of the amplification products. The fourth nucleic acid probe detects the correct nucleic acid sequence of the amplification product to reduce false positives.
Embodiments of the present method can be used to quantitate the amount of first nucleic acids having a target sequence. The number of cycles and the amount of signals generated by the amplification product relate to the amount of first nucleic acid having a target sequence initially present.
One embodiment of the present method features a support with many sets of second and third nucleic acids, with each set directed to a different first nucleic acid. Preferably, each set of second and third nucleic acids are positioned in discrete areas of the support. Each support may comprise a plurality of sets to a plurality of first nucleic acids and targets. Preferably, at least one set has a second and third nucleic acid having a nonsense sequence, which nonsense sequence is not intended under hybridization and amplification conditions to generate an amplification product as a negative control. Preferably, at least one set has a second and third nucleic acid having a sequence which is universally present in almost all samples, or is directed to a nucleic acid sequence present in the sample as a positive control.
Embodiments of the present method are also useful for mapping large nucleic acids. One embodiment of the present invention features sets of second and third nucleic acids. Each set is directed to a first nucleic acid which first nucleic acid is part of a large nucleic acid. The sets of second and third nucleic acids generate sets of first and second amplification products. The sets of first and second amplification products span overlapping sequences of the large nucleic acid. As used herein, xe2x80x9coverlappingxe2x80x9d refers to two nucleic acids having at least one identical nucleotide sequence directed to an area of nucleic acid from which they were derived or to which they are intended to hybridize. These overlapping sequences can be correlated to produce a map of the large nucleic acid. The present invention can be used to replace the use of sequence tag sites by using an array of amplification products.
One embodiment of the present invention features a method which is capable of forming a precipitate or agglutination product in the presence of a first nucleic acid having a target sequence. This method features a first support having a second nucleic acid and a second support having a third nucleic acid. The method comprises forming an immersion product of the first and second support with a sample potentially containing the first nucleic acid. Next, hybridization conditions are imposed on the immersion product to form a first hybridization product comprising the first nucleic acid and the second nucleic acid. Next, amplification conditions are imposed on the hybridization product to form a first amplification product. The third nucleic acid has a sequence identical to a second target sequence. That is, the third nucleic acid is complementary to at least a section of the first amplification product. The method further comprises forming a second hybridization product comprising the third nucleic acid and the first amplification product extending from the first support. This second hybridization product can promote an agglutination or precipitation of the immersion product.
Preferably, this hybridization product is further stabilized by forming a second amplification product comprising a nucleic acid extending from the third nucleic acid complementary to the first amplification product.
Preferably, the immersion product comprises a suspension of supports. Each support having a plurality of second and third nucleic acids which upon cycling through hybridization, denaturation and amplification conditions form an agglutination product.
The present method is ideally suited for applications utilizing carboxylated latex particles, plastic or glass beads which can precipitate from solutions upon the formation of a second hybridization product or a second amplification product.
A further embodiment of the present invention features an article of manufacture for forming an amplification product in the presence of a first nucleic acid having a target sequence. The article of manufacture comprises a support having a second nucleic acid having a nucleotide sequence complementary to the target sequence, which second nucleic acid is covalently bound to the support. The support is capable of forming an immersion product with a sample potentially containing the first nucleic acids, undergoing hybridization conditions and undergoing amplification conditions to form a first hybridization product and a first amplification product in the presence of the first nucleic acid. The first amplification product extends from the second nucleic acid and is complementary to the first nucleic acid.
Preferably, the article of manufacture further comprises a third nucleic acid in which the third nucleic acid is complementary to the first amplification product. The third nucleic acid is capable of forming a second hybridization product with a target sequence of the first amplification upon imposition of hybridization conditions. That is, the third nucleic acid is homologous to a second target sequence of the first nucleic acid. Preferably, the third nucleic acid is capable of priming a reaction to form a second amplification product.
Preferably, the second and third nucleic acid have a spaced relationship. The positions are separated by a distance less than the length of the first target sequence and the second target sequence of the first nucleic acid. In the alternative, the second and third nucleotides are randomly immobilized in a concentration or density on the support such that first and second nucleic acids have a spaced relationship.
Preferably, each set occupies a defined region of the support to form a pixel-like area. The amplification product occurs in the defined region and is termed the test site or test pixel.
Preferably, in order to effect mapping of a first nucleic acid, the support has a plurality of sets of second and third nucleic acids. Each set corresponds to a first nucleic acid which first nucleic acid is part of a large nucleic acid. The first nucleic acid for each set preferably has overlapping sequences to allow mapping of the first nucleic acid by matching the overlapping area.
Embodiments of the present invention are well suited for automation. A further embodiment of the present invention features an instrument for forming an amplification product in the presence of a first nucleic acid having a target sequence. The instrument comprises means for receiving a support having a second nucleic acid having a nucleotide sequence complementary to the target sequence. The second nucleic acid is covalently bound to the support. The instrument further comprises means for forming an immersion product comprising the support and the sample. The instrument further comprises means for imposing hybridization conditions on the immersion product for forming a hybridization product in the presence of the first nucleic acid. The instrument further comprises means for imposing amplification conditions on the hybridization product, if formed, to form an amplification product. The formation of the amplification product can be related to the presence of the first nucleic acid and the target sequence.
Means for forming an immersion product may comprise apparatus for depositing a sample on a support containing the second nucleic acid or means for placing the solid support within a containment vessel containing the sample.
Means for imposing hybridization conditions comprises devices such as dispensing orifices, pipettes and the like for placing suitable buffers with the immersion product and temperature controls to effect hybridization of nucleic acid.
Means for imposing amplification conditions comprises devices such as dispensing orifices, pipettes and the like for placing enzymes and reagents for extending nucleic acid or duplicating nucleic acid. Typical reagents include polymerases, nucleotides, buffers and the like. The conditions for imposing hybridization conditions and amplification conditions are well known to individuals skilled in the art.
Thus, the present invention features methods, devices and articles of manufacture for the detection of nucleic acid having a particular target sequence without using solution based primer sets. The present invention facilitates the performance of simultaneous target amplification reactions, greatly shortening the time to generate data necessary to map nucleic acids. The absence of an external reaction chamber and a gel base system for the analysis of amplified products greatly facilitates the analysis of the assay results.
These and other features will become apparent from the drawings and the detailed description which follow which, by way of example, without limitation, describe preferred embodiments of the present invention.